Using arterial-venous analysis to characterize cancer metabolic consumption in patients.

Understanding tumor metabolism holds the promise of new insights into cancer biology, diagnosis and treatment. To assess human cancer metabolism, here we report a method to collect intra-operative samples of blood from an artery directly upstream and a vein directly downstream of a brain tumor, as well as samples from dorsal pedal veins of the same patients. After performing targeted metabolomic analysis, we characterize the metabolites consumed and produced by gliomas in vivo by comparing the arterial supply and venous drainage. N-acetylornithine, D-glucose, putrescine, and L-acetylcarnitine are consumed in relatively large amounts by gliomas. Conversely, L-glutamine, agmatine, and uridine 5-monophosphate are produced in relatively large amounts by gliomas. Further we verify that D-2-hydroxyglutarate (D-2HG) is high in venous plasma from patients with isocitrate dehydrogenases1 (IDH1) mutations. Through these paired comparisons, we can exclude the interpatient variation that is present in plasma samples usually taken from the cubital vein.

New, simple and sensitive HPTLC method for simultaneous determination of anti-hepatitis C sofosbuvir and ledipasvir in rabbit plasma.

Sofosbuvir (SOF) and ledipasvir (LDS) represent anti-hepatitis C binary mixture. Herein, a fast high-performance thin-layer chromatography (HPTLC) method was developed, validated and applied for simultaneous determination of SOF and LDS in biological matrix. An innovative strategy was designed which based on coupling dual wavelength detection with HPTLC. This strategy enabled sensitive, specific, high sample throughput and cost-effective determination of the SOF-LDS binary mixture. The developed HPTLC procedure is based on a simple liquid-liquid extraction, enrichment of the analytes and subsequent separation with UV detection. Separations were performed on HPTLC silica gel 60 F254 aluminum plates with a mobile phase consisting of ethyl acetate-glacial acetic acid (100:5, v/v). The Rf values for SOF and LDS were 0.62 and 0.30, respectively. Dual wavelength scanning was carried out in the absorbance mode at 265 and 327 nm for SOF and LDS, respectively. The linear ranges were 40-640 and 9-144 ng/band for SOF and LDS, respectively with correlation coefficients of 0.9998. The detection limits were 10.61 and 2.54 ng/band and the quantitation limits were 32.14 and 7.70 ng/band for SOF and LDS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in rabbit plasma with good percentage recovery (95.68-103.26%). Validation parameters were assessed according to ICH guidelines. The proposed method represents a simple, high sample throughput and economic alternative to the already existing more complicated reported LC-MS/MS techniques. The method would afford an efficient tool for therapeutic drug monitoring and bioavailability studies of SOF and LDS.

A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma.

A rapid and stereospecific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the separation and determination of PSI-7851 diastereomers in human K2EDTA plasma has been developed. The analytical method involves direct protein precipitation with acetonitrile, followed by separation of the diastereomers on a Luna C18 column, positive mode electrospray ionization and selected reaction monitoring mode mass spectrometry detection. The mobile phase composition and pH were investigated for the resolution of the two diastereomers of PSI-7851. The optimized method showed good resolution (R(s) = 4.8) within short analysis time (approximately 8 min). The assay range was 5-2500 ng/mL for both diastereomers using a 1/x² weighted linear regression analysis for standard curve fitting. Replicate sample analysis indicated that intra- and inter-day accuracy and precision were within ±15.0%. The recovery of diastereomers from human plasma was greater than 85% and no significant matrix effect was observed. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies.

[Activity of pyrimidine 5'nucleotidase (py5'Nd) of erythrocytes in essential hypertension]. / Aktywnosc nukleotydazy 5'pirymidynowej (Py5'Nd) krwinek czerwonych w nadcisnieniu tetniczym pierwotnym.

Activity of pyrimidine 5'nucleotidase was measured in hemolysate of erythrocytes of healthy persons and patients with essential hypertension. Cytidine 5'monophosphate (CMP) and uridine 5'monophosphate (UMP) were used as the substrates for evaluation of activity of so called I-isoenzyme and uridine 3'monophosphate (U3'MP) was used as a substrate for the II isoenzyme of Py5'Nd. It was found that the activity of Py5'Nd I was lower in hypertensives (26.8 mU/gHb (CMP)) and 69.3 mU/gHb (UMP) in comparison with normotensives (62.3 and 117.4 mU/gHb respectively) (p < 0.05). The activity of Py5'Nd II did not differ between studied groups. Possible metabolic consequences of decreased activity of Py5'Nd are discussed.

Metabolic studies of uridine in rats with DOCA-salt hypertension and on high sodium diet.

1. The steady-state metabolic clearance and calculated secretion rate of the pyrimidine nucleoside uridine were studied by equilibrium infusion in normal rats, rats on a high sodium diet, rats made hypertensive by subcutaneous injection of deoxycorticosterone acetate (DOCA), unilateral nephrectomy and high sodium drinking fluid, and two control groups of rats for the hypertensive group. 2. Basal plasma uridine concentration in DOCA-salt hypertension rats was found to be significantly reduced to 3.99 +/- 0.31 mumol/L (mean +/- s.e.m.) compared with control rats (11.98 +/- 1.64 mumol/L). Metabolic clearance (MCR) in DOCA-salt hypertensive rats was significantly raised (200.54 +/- 10.77 mL/kg per min) compared with control rats (65.17 +/- 1.99 mL/kg per min). No difference was found in plasma uridine concentration and MCR among the other two control groups and high sodium diet rats. Calculated secretion rate was unchanged in all animals. No significant differences were found between different groups of rats in blood pressure responses to uridine. 3. The raised metabolic clearance and reduced plasma uridine concentration in DOCA-salt hypertension may be consistent with increased intracellular transport and phosphorylation of uridine to the physiologically active compound uridine monophosphate (UMP) which would lead to arteriolar constriction, hypertension and natriuresis. The results contrast with those in humans with extracellular fluid (ECF) expansion from endstage renal failure and rats with one-kidney, one-clip (1K1C) hypertension but are not due to the pharmacological effects of deoxycorticosterone. The difference may be due to the haemodynamic consequences of reduced renal perfusion pressure or reduced renal mass compared with DOCA-salt model.

Metabolism of uridine in expanded extracellular volume states.

1. Uridine and uridine monophosphate (UMP) are natriuretic and a vasopressor in intact rats. In deoxycorticosterone acetate (DOCA)-salt hypertensive rats metabolic clearance rate (MCR) of uridine is raised and basal plasma uridine diminished, suggesting that metabolism of uridine is linked to changes in extracellular space. 2. Plasma uridine concentration was raised in 38 patients with chronic renal failure compared with age- and sex-matched healthy controls (8.49 mumol/L, 4.37-13.74 mumol/L median, interquartile range, and 2.64 mumol/L 2.51-2.74 mumol/L, respectively, P < 0.001). Plasma uridine was significantly diminished after isotonic fluid removal by ultrafiltration (UF) from 7.25 mumol/L (3.7-11.08) to 5.07 mumol/L (3.3-8.3), P < 0.001, whereas concentration of marker solutes urea and creatinine remained unchanged. During haemodialysis (HD), plasma uridine fell significantly from its pre-HD level. 3. In an animal model of expanded extracellular space the one-kidney, one-clip rat, plasma uridine was significantly higher (20.56 +/- 1.19 mumol/L, P < 0.01) and MCR diminished (34.93 +/- 3.44 mL/kg per min, P < 0.01) compared with sham-operated animals (plasma uridine 12.14 +/- 1.07 and MCR 53.59 +/- 4.11 mL/kg per min). Uridine or UMP did not inhibit Na+, K(+)-ATPase in either of the two assay systems. 4. It was concluded that catabolism of uridine is reduced by extracellular expansion and probably increased by volume reduction by UF.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of erythrocyte nucleotides in pyrimidine 5'-nucleotidase deficiency.

In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion exchange chromatography on Dowex formate columns using a linear ammonium formate elution gradient. In contradistinction to normal erythrocytes, adenine nucleotides accounted for only 32% of the nucleotide pool. The remainder consisted of 50% cytidine and 16% uridine nucleotides. The remaining 2% was not identified. The most abundant compound appeared to be UDP glucose whilst high levels of CTP, CMP and an unidentified cytidine compound less polar than CMP accounted for most of the cytidine nucleotide pool. The possibility that the abnormal nucleotides were due to an elevated reticulocyte count was excluded and it was also shown that erythrocytes from subjects heterozygous for pyrimidine 5'-nucleotidase deficiency did not have detectable levels of the abnormal nucleotides.

Serum 'uracil plus uridine' levels in normal subjects and their possible significance.

A microbiological method for the assay of uracil is described. The growth of the test organism is supported by uracil and also by uridine but not by uridylic acid. The method therefore measures uracil and uridine together. The ;uracil + uridine' level, expressed as uracil, has been measured in blood from 144 normal subjects ranging in age from cord blood to the eighth decade. The mean level of 22 mu mol/l (0.25 mg%) in cord blood decreases to 15 mumol/l (0.17 mg%) in adults over the age of 20. There is no difference between the sexes. Uracil is of interest because (a) it is a constituent base of RNA, (b) it is the precursor of two of the bases thymine and cytosine that enter into the composition of DNA, and (c) under certain circumstances it has mutagenic properties. The last is dependent upon the existence of two tautomeric forms of uracil, the common keto form which pairs normally with adenine and the rare enol form which pairs with guanine. A mistake in base pairing which allows uracil in its enol form to enter the DNA molecule and pair with guanine can result in a G = C --> A = T base transition in the DNA molecule. The molecular mechanism involved as well as the possible bearing on somatic mutation are discussed.